Tailored Viral-like Particles as Drivers of Medical Breakthroughs.

Despite the recognized potential of nanoparticles, only a few formulations have progressed to clinical trials, and an even smaller number have been approved by the regulatory authorities and marketed. Virus-like particles (VLPs) have emerged as promising alternatives to conventional nanoparticles due to their safety, biocompatibility, immunogenicity, structural stability, scalability, and versatility. Furthermore, VLPs can be surface-functionalized with small molecules to improve circulation half-life and target specificity. Through the functionalization and coating of VLPs, it is possible to optimize the response properties to a given stimulus, such as heat, pH, an alternating magnetic field, or even enzymes. Surface functionalization can also modulate other properties, such as biocompatibility, stability, and specificity, deeming VLPs as potential vaccine candidates or delivery systems. This review aims to address the different types of surface functionalization of VLPs, highlighting the more recent cutting-edge technologies that have been explored for the design of tailored VLPs, their importance, and their consequent applicability in the medical field.

Soil actinobacteria inhibit antagonistic fungi of leafcutter ant colonies.

Founder females of the leaf-cutting ant species Atta sexdens experience high mortality during the founding and establishment of their colonies. The foundation site is crucial for the success of a new colony. In this study, we isolated and identified actinobacteria from fungus garden chambers of A. sexdens colony growth in soils from (1) forested areas without leafcutter ant nests and (2) open ground areas close to leafcutter ant nests. The inhibitory effect of these isolates on pathogenic fungi and the mutualistic fungus cultivated by leafcutter ants was evaluated. The 16S rRNA gene sequences were employed to identify nine selected actinobacteria species found in the soil: Streptomyces (6), Nocardia (2), and Kitasatospora (1). One Streptomyces and one Kitasatospora isolate inhibited all the tested fungi. Since there is no evidence of actinobacteria cultivation in the workers' cuticle of the Atta genus, our results corroborate the hypothesis that these workers may establish temporary adaptive symbiosis with soil microorganisms that produce antibiotic substances, living in some parts of their nest, or even inside their bodies. Pathogenic fungi are a risk factor that can be controlled by actinobacteria metabolites from soils, with minimal energy cost to the colony.

Integrated in Silico and Experimental Approach towards the Design of a Novel Recombinant Protein Containing an Anti-HER2 scFv.

Biological therapies, such as recombinant proteins, are nowadays amongst the most promising approaches towards precision medicine. One of the most innovative methodologies currently available aimed at improving the production yield of recombinant proteins with minimization of costs relies on the combination of in silico studies to predict and deepen the understanding of the modified proteins with an experimental approach. The work described herein aims at the design and production of a biomimetic vector containing the single-chain variable domain fragment (scFv) of an anti-HER2 antibody fragment as a targeting motif fused with HIV gp41. Molecular modeling and docking studies were performed to develop the recombinant protein sequence. Subsequently, the DNA plasmid was produced and HEK-293T cells were transfected to evaluate the designed vector. The obtained results demonstrated that the plasmid construction is robust and can be expressed in the selected cell line. The multidisciplinary integrated in silico and experimental strategy adopted for the construction of a recombinant protein which can be used in HER2+-targeted therapy paves the way towards the production of other therapeutic proteins in a more cost-effective way.

Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6.

Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared "classical" fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH2) and the novel peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the "classical" method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the "classical" SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a "working day" (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3.

Understanding the Binding Specificity of G-Protein Coupled Receptors toward G-Proteins and Arrestins: Application to the Dopamine Receptor Family.

G-Protein coupled receptors (GPCRs) are involved in a myriad of pathways key for human physiology through the formation of complexes with intracellular partners such as G-proteins and arrestins (Arrs). However, the structural and dynamical determinants of these complexes are still largely unknown. Herein, we developed a computational big-data pipeline that enables the structural characterization of GPCR complexes with no available structure. This pipeline was used to study a well-known group of catecholamine receptors, the human dopamine receptor (DXR) family and its complexes, producing novel insights into the physiological properties of these important drug targets. A detailed description of the protein interfaces of all members of the DXR family (D1R, D2R, D3R, D4R, and D5R) and the corresponding protein interfaces of their binding partners (Arrs: Arr2 and Arr3; G-proteins: Gi1, Gi2, Gi3, Go, Gob, Gq, Gslo, Gssh, Gt2, and Gz) was generated. To produce reliable structures of the DXR family in complex with either G-proteins or Arrs, we performed homology modeling using as templates the structures of the ß2-adrenergic receptor (ß2AR) bound to Gs, the rhodopsin bound to Gi, and the recently acquired neurotensin receptor-1 (NTSR1) and muscarinic 2 receptor (M2R) bound to arrestin (Arr). Among others, the work demonstrated that the three partner groups, Arrs and Gs- and Gi-proteins, are all structurally and dynamically distinct. Additionally, it was revealed the involvement of different structural motifs in G-protein selective coupling between D1- and D2-like receptors. Having constructed and analyzed 50 models involving DXR, this work represents an unprecedented large-scale analysis of GPCR-intracellular partner interface determinants. All data is available at www.moreiralab.com/resources/dxr.

The Central Role of Non-Structural Protein 1 (NS1) in Influenza Biology and Infection.

Influenza (flu) is a contagious viral disease, which targets the human respiratory tract and spreads throughout the world each year. Every year, influenza infects around 10% of the world population and between 290,000 and 650,000 people die from it according to the World Health Organization (WHO). Influenza viruses belong to the Orthomyxoviridae family and have a negative sense eight-segment single-stranded RNA genome that encodes 11 different proteins. The only control over influenza seasonal epidemic outbreaks around the world are vaccines, annually updated according to viral strains in circulation, but, because of high rates of mutation and recurrent genetic assortment, new viral strains of influenza are constantly emerging, increasing the likelihood of pandemics. Vaccination effectiveness is limited, calling for new preventive and therapeutic approaches and a better understanding of the virus-host interactions. In particular, grasping the role of influenza non-structural protein 1 (NS1) and related known interactions in the host cell is pivotal to better understand the mechanisms of virus infection and replication, and thus propose more effective antiviral approaches. In this review, we assess the structure of NS1, its dynamics, and multiple functions and interactions, to highlight the central role of this protein in viral biology and its potential use as an effective therapeutic target to tackle seasonal and pandemic influenza.

Root distribution and its association with bean growth habit.

Associations between root distribution and bean growth habit may contribute to the selection of genotypes adapted to restrictive environments. The present work aimed to relate and compare root distribution with the growth habit in beans. 10 bean genotypes of different growth habits (I, II and III) were evaluated for root distribution in two agricultural years (2014/15 and 2015/16). The genotypes responded similarly for the trait root distribution throughout the agricultural years, without any simple effect of the genotype x year interaction. The factors genotype and years were significant for the trait. The genotypes of a determinate habit showed significant differences compared to other genotypes (II and III), which were ascribed to their poor performance in the average of years. They include the Carioca Precoce, which showed a behavior similar to the other habits. It could be considered a "plastic" genotype under restrictive conditions. The contrasts revealed significant differences between the growth habits II vs I (2.87) and III vs I (3.64) for root distribution. The differences were also significant for grain yield. Thus, genotypes of indeterminate growth habit show greater root distribution than those of a determinate habit, regardless of the agricultural years. Therefore, they are promising for use in blocks of crosses, when the purpose is the selection of cultivars adapted to low input environments.

Less Exploited GPCRs in Precision Medicine: Targets for Molecular Imaging and Theranostics.

Precision medicine relies on individually tailored therapeutic intervention taking into account individual variability. It is strongly dependent on the availability of target-specific drugs and/or imaging agents that recognize molecular targets and patient-specific disease mechanisms. The most sensitive molecular imaging modalities, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), rely on the interaction between an imaging radioprobe and a target. Moreover, the use of target-specific molecular tools for both diagnostics and therapy, theranostic agents, represent an established methodology in nuclear medicine that is assuming an increasingly important role in precision medicine. The design of innovative imaging and/or theranostic agents is key for further accomplishments in the field. G-protein-coupled receptors (GPCRs), apart from being highly relevant drug targets, have also been largely exploited as molecular targets for non-invasive imaging and/or systemic radiotherapy of various diseases. Herein, we will discuss recent efforts towards the development of innovative imaging and/or theranostic agents targeting selected emergent GPCRs, namely the Frizzled receptor (FZD), Ghrelin receptor (GHSR-1a), G protein-coupled estrogen receptor (GPER), and Sphingosine-1-phosphate receptor (S1PR). The pharmacological and clinical relevance will be highlighted, giving particular attention to the studies on the synthesis and characterization of targeted molecular imaging agents, biological evaluation, and potential clinical applications in oncology and non-oncology diseases. Whenever relevant, supporting computational studies will be also discussed.

Recovery of phenolic compounds from multi-component solution by a synthesized activated carbon using resorcinol and formaldehyde.

The adsorption of four phenolic compounds (gallic acid, protocatechuic acid, vanillic acid and syringic acid) is investigated using a synthesized mesoporous carbon on both single and multi-component synthetic solutions. Some correlation of the adsorption capacity of the carbon and the nature of adsorbate could be made, except for gallic acid whose concentration decrease seems to be not exclusively due to adsorption but also to polymerization reaction. In the multi-component mixture, negative effects in the adsorption capacity are observed probably due to competition for the active centers of the adsorbent surface. In desorption studies, ethanol presents better performance than water and acetonitrile. Vanillic acid is the compound with the higher adsorption and interestingly it is then possible to desorb a relatively high amount of it from the adsorbent, which may represent a possibility for a selective recovery of vanillic acid. These results present a potential way to treat the wastewater from the cork industry.

A Machine Learning Approach for Hot-Spot Detection at Protein-Protein Interfaces.

Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS) in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML) techniques. Our model is trained on a large number of complexes and on a significantly larger number of different structural- and evolutionary sequence-based features. In particular, we added interface size, type of interaction between residues at the interface of the complex, number of different types of residues at the interface and the Position-Specific Scoring Matrix (PSSM), for a total of 79 features. We used twenty-seven algorithms from a simple linear-based function to support-vector machine models with different cost functions. The best model was achieved by the use of the conditional inference random forest (c-forest) algorithm with a dataset pre-processed by the normalization of features and with up-sampling of the minor class. The method has an overall accuracy of 0.80, an F1-score of 0.73, a sensitivity of 0.76 and a specificity of 0.82 for the independent test set.

Mouse model for sublethal Leptospira interrogans infection.

Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1ß, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4(+) and double-negative T cells (not CD8(+) cells) and that CD4(+) T cells acquired a CD44(high) CD62L(low) effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection).

Reservoir targeted vaccine against Borrelia burgdorferi: a new strategy to prevent Lyme disease transmission.

A high prevalence of infection with Borrelia burgdorferi in ixodid ticks is correlated with a high incidence of Lyme disease. The transmission of B. burgdorferi to humans can be disrupted by targeting 2 key elements in its enzootic cycle: the reservoir host and the tick vector. In a prospective 5-year field trial, we show that oral vaccination of wild white-footed mice resulted in outer surface protein A-specific seropositivity that led to reductions of 23% and 76% in the nymphal infection prevalence in a cumulative, time-dependent manner (2 and 5 years, respectively), whereas the proportion of infected ticks recovered from control plots varied randomly over time. Significant decreases in tick infection prevalence were observed within 3 years of vaccine deployment. Implementation of such a long-term public health measure could substantially reduce the risk of human exposure to Lyme disease.

Oral immunization with Escherichia coli expressing a lipidated form of LigA protects hamsters against challenge with Leptospira interrogans serovar Copenhageni.

Leptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals that harbor leptospires in their renal tubules and shed the bacteria in their urine. Leptospira interrogans serovar Copenhageni transmitted from Rattus norvegicus to humans is the most prevalent cause of urban leptospirosis. We examined L. interrogans LigA, domains 7 to 13 (LigA7-13), as an oral vaccine delivered by Escherichia coli as a lipidated, membrane-associated protein. The efficacy of the vaccine was evaluated in a susceptible hamster model in terms of the humoral immune response and survival from leptospiral challenge. Four weeks of oral administration of live E. coli expressing LigA7-13 improved survival from intraperitoneal (i.p.) and intradermal (i.d.) challenge by L. interrogans serovar Copenhageni strain Fiocruz L1-130 in Golden Syrian hamsters. Immunization with E. coli expressing LigA7-13 resulted in a systemic antibody response, and a significant LigA7-13 IgG level after the first 2 weeks of immunization was completely predictive of survival 28 days after challenge. As in previous LigA vaccine studies, all immunized hamsters that survived infection had renal leptospiral colonization and histopathological changes. In summary, an oral LigA-based vaccine improved survival from leptospiral challenge by either the i.p. or i.d. route.

Presence of fimH, mrkD, and irp2 virulence genes in KPC-2-producing Klebsiella pneumoniae isolates in Recife-PE, Brazil.

Klebsiella pneumoniae strains can produce different virulence factors, such as fimbrial adhesins and siderophores, which are important in the colonization and development of the infection. The aims of this study were to determine the occurrence of fimH, mrkD, and irp2 virulence genes in 22 KPC-2-producing K. pneumoniae isolates as well as 22 not producing-KPC isolates, from patients from different hospitals in Recife-PE, Brazil, and also to analyze the clonal relationship of the isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The genes were detected by PCR and DNA sequencing. The bla KPC-2 gene was identified in 22 KPC-positive isolates. On analyzing the antimicrobial susceptibility profile of the isolates, it was detected that polymyxin and amikacin were the antimicrobials of best activity against K. pneumoniae. On the other hand, five isolates exhibited resistance to polymyxin. In the KPC-positive group, was observed a high rate of resistance to cephalosporins, followed by carbapenems. Molecular typing by ERIC-PCR detected 38 genetic profiles, demonstrating a multiclonal spread of the isolates analyzed. It was observed that the virulence genes irp2, mrkD, and fimH were seen to have together a higher frequency in the KPC-positive group. The accumulation of virulence genes of KPC-positive K. pneumoniae isolates, observed in this study, along with the multi-resistance impose significant therapeutic limitations on the treatment of infections caused by K. pneumoniae.

Investigating the Antimicrobial Potential of 560 Compounds from the Pandemic Response Box and COVID Box against Resistant Gram-Negative Bacteria.

Antimicrobial resistance (AMR) has emerged as a significant threat to public health, particularly in infections caused by critically important Gram-negative bacteria. The development of novel antibiotics has its limitations, and therefore it is crucial to explore alternative strategies to effectively combat infections with resistant pathogens. In this context, the present study investigated the antibacterial potency of 560 compounds against the multidrug-resistant (MDR) strains of Klebsiella pneumoniae and Serratia marcescens. The evaluated compounds were selected from the Pandemic Response Box (PRB) and COVID Box (CB) and subjected to assays to determine the inhibitory concentration (IC), minimum bactericidal concentration (MBC), and biofilm formation. Further, the effects of these compounds on membrane integrity were assessed through protein quantification. Several of the evaluated compounds, including fusidic acid, MMV1580853, and MMV1634399, exhibited a significant reduction in biofilm formation and growth in K. pneumoniae. Trimethoprim exhibited potential against S. marcescens. The IC values of the compounds indicated significant microbial growth inhibition at various concentrations. These findings underscore the potency of the existing antibiotics and novel compounds in combating the MDR strains of bacteria. The importance of reconsidering the known antibiotics and utilizing drug repositioning strategies to address the increasing risk of AMR is highlighted.

The World of GPCR dimers - Mapping dopamine receptor D2 homodimers in different activation states and configuration arrangements.

G protein-coupled receptors (GPCRs) are known to dimerize, but the molecular and structural basis of GPCR dimers is not well understood. In this study, we developed a computational framework to generate models of symmetric and asymmetric GPCR dimers using different monomer activation states and identified their most likely interfaces with molecular details. We chose the dopamine receptor D2 (D2R) homodimer as a case study because of its biological relevance and the availability of structural information. Our results showed that transmembrane domains 4 and 5 (TM4 and TM5) are mostly found at the dimer interface of the D2R dimer and that these interfaces have a subset of key residues that are mostly nonpolar from TM4 and TM5, which was in line with experimental studies. In addition, TM2 and TM3 appear to be relevant for D2R dimers. In some cases, the inactive configuration is unaffected by the partnered protomer, whereas in others, the active protomer adopts the properties of an inactive receptor. Additionally, the ß-arrestin configuration displayed the properties of an active receptor in the absence of an agonist, suggesting that a switch to another meta-state during dimerization occurred. Our findings are consistent with the experimental data, and this method can be adapted to study heterodimers and potentially extended to include additional proteins such as G proteins or ß-arrestins. In summary, this approach provides insight into the impact of the conformational status of partnered protomers on the overall quaternary GPCR macromolecular structure and dynamics.

Impact of concerning excipients on animal safety: insights for veterinary pharmacotherapy and regulatory considerations.

OBJECTIVES: Veterinarians and pharmacists are familiar with the efficacy and safety aspects attributed to active pharmaceutical ingredients included in medicines, but they are rarely concerned with the safety of excipients present in medicines. Although generally recognized as safe, excipients are not chemically inert and may produce adverse events in certain animal populations. This review aims to present excipients of concern to these populations and highlight their relevance for rational veterinary pharmacotherapy. EVIDENCE ACQUISITION: A comprehensive review of the literature about the existence of adverse reactions in animals caused by pharmaceutical excipients was carried out based on an exploratory study. An overview of the correct conditions of use and safety of these excipients has also been provided, with information about their function, the proportion in which they are included in the different pharmaceutical dosage forms and the usual routes of administration. RESULTS: We identified 18 excipients considered of concern due to their potential to cause harm to the health of specific animal populations: bentonite, benzalkonium chloride, benzoic acid, benzyl alcohol, ethanol, lactose, mannitol, mineral oil, monosodium glutamate, polyethylene glycol, polysorbate, propylene glycol, sodium benzoate, sodium carboxymethylcellulose, sodium lauryl sulfate, sulfites, polyoxyethylene castor oil derivatives, and xylitol. Among the 135 manuscripts listed, only 24 referred to studies in which the substances were correctly evaluated as excipients. CONCLUSIONS: Based on the information presented in this review, the authors hope to draw the attention of professionals involved in veterinary pharmacotherapy to the existence of excipients of concern in medicines. This information contributes to rational veterinary pharmacotherapy and supports veterinary pharmacovigilance actions. We hope to shed light on the subject and encourage studies and new manuscripts that address the safety of pharmaceutical excipients to the animal population.

Enhanced neuronal differentiation by dynamic piezoelectric stimulation.

Electroactive smart materials play an important role for tissue regenerative applications. Poly(vinylidene fluoride) (PVDF) is a specific subtype of piezoelectric electroactive material that generates electrical potential upon mechanical stimulation. This work focuses on the application of piezoelectric PVDF films for neural differentiation. Human neural precursor cells (hNPCs) are cultured on piezoelectric poled and non-poled ß-PVDF films with or without a pre-coating step of poly-d-lysine and laminin (PDL/L). Subsequently, hNPCs differentiation into the neuronal lineage is assessed (MAP2+ and DCX+ ) under static or dynamic (piezoelectric stimulation) culture conditions. The results demonstrate that poled and coated ß-PVDF films induce neuronal differentiation under static culture conditions which is further enhanced with mechanical stimulation. In silico calculations of the electrostatic potential of different domains of laminin, highlight the high polarity of those domains, which shows a clear preference to interact with the varying surface electric field of the piezoelectric material under mechanical stimulation. These interactions might explain the higher neuronal differentiation induced by poled ß-PVDF films pre-coated with PDL/L under dynamic conditions. Our results suggest that electromechanical stimuli, such as the ones induced by piezoelectric ß-PVDF films, are suitable to promote neuronal differentiation and hold great promise for the development of neuroregenerative therapies.

Construction of HER2-Specific HIV-1-Based VLPs.

Virus-like particles (VLPs) are nanoplatforms comprised of one or more viral proteins with the capacity to self-assemble without viral genetic material. VLPs arise as promising nanoparticles (NPs) that can be exploited as vaccines, as drug delivery vehicles or as carriers of imaging agents. Engineered antibody constructs, namely single-chain variable fragments (scFv), have been explored as relevant molecules to direct NPs to their target. A vector containing the scFv of an antibody, aimed at the human epidermal growth factor receptor 2 (HER2) and fused to the human immunodeficiency virus (HIV) protein gp41, was previously constructed. The work herein describes the early results concerning the production and the characterization of HIV-1-based VLPs expressing this protein, which could function as potential non-toxic tools for transporting drugs and/or imaging agents.

How promising are HIV-1-based virus-like particles for medical applications.

New approaches aimed at identifying patient-specific drug targets and addressing unmet clinical needs in the framework of precision medicine are a strong motivation for researchers worldwide. As scientists learn more about proteins that drive known diseases, they are better able to design promising therapeutic approaches to target those proteins. The field of nanotechnology has been extensively explored in the past years, and nanoparticles (NPs) have emerged as promising systems for target-specific delivery of drugs. Virus-like particles (VLPs) arise as auspicious NPs due to their intrinsic properties. The lack of viral genetic material and the inability to replicate, together with tropism conservation and antigenicity characteristic of the native virus prompted extensive interest in their use as vaccines or as delivery systems for therapeutic and/or imaging agents. Owing to its simplicity and non-complex structure, one of the viruses currently under study for the construction of VLPs is the human immunodeficiency virus type 1 (HIV-1). Typically, HIV-1-based VLPs are used for antibody discovery, vaccines, diagnostic reagent development and protein-based assays. This review will be centered on the use of HIV-1-based VLPs and their potential biomedical applications.